Flow cytometers are used in life science research for quantitative assays of large populations of biological cells and particles. A beam of light (typically of a single wavelength) is directed onto a hydrodynamically focused stream of fluid. The fluid stream typically includes a fluid carrier or “sheath” and a core fluid including a plurality of particles. The fluid stream generally permits one particle to pass through the light beam at a time. A number of detectors can be aimed at the point where the stream passes through the light beam. For example, a detector can be positioned in line with the light beam to detect forward scatter and one or more detectors can be positioned perpendicular to the light beam to detect side scatter. The particles can contain fluorescent components, and one or more fluorescent detectors can be used to detect a resulting fluorescence signal. Each suspended particle passing through the light beam scatters the light in some way and/or a fluorescent component on the particle may fluoresce light, e.g., at a lower frequency than the light source. Scattered and fluorescent light can be detected and analyzed.
The signals detected from a flow cytometer can be used to characterize the physical and/or chemical structure of the particles. For example, the forward scattered light can be correlated with a cell volume, and the side scattered light may be correlated with the shape or other inner features of the particle. The scattered and/or fluorescence signals are acquired by detectors that allow fast signal acquisition (e.g., thousands of cells per second) and rapid data analysis for a large cell population. For example, the cells can be classified in a multi-dimensional feature space defined by various fluorescence signals, forward scatter signals, and/or side scatter signals.
More recently, imaging flow cytometers are available in which a CCD camera is used to record bright-field, dark field, and fluorescent images. These imaging flow cytometers use microscope techniques to acquire two dimensional images from each interrogated cell for analysis of features at a rate of up to about 100 cells per second. However, these techniques rely on conventional fluorescence or bright-field microscopy in which the resulting images are non-diffractional and inherently two dimensional replicas of the three dimensional cell structure (with the third dimension being compressed into a “focal depth”). Although these two dimensional images can be analyzed with pattern recognition algorithms, automated analysis with existing pattern recognition algorithms is complex, labor intensive, and challenging at least because of the two dimensional nature of the image.
Confocal imaging techniques have been used in non-flow applications to acquire multiple two dimensional non-diffraction images of very short focal depth and stack them along the third dimension to provide a three dimensional construction. However, this technique typically requires multiple images and, therefore, these confocal imaging techniques are generally not compatible with an imaging flow cytometer in which the particles are moving relatively rapidly.
In addition, the high flow speeds and poor signal to noise ratios in conventional flow cytometers may limit the amount of information that can be extracted from the scattering and/or fluorescence signals.